Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition

Cyclosporine A (CsA), a widely used immunosuppressant shows cytotoxic effects by either inducing apoptosis or redirecting the cell towards non-apoptotic cell death. However, there still remains a lacuna in understanding the mechanism of CsA induced non-apoptotic cell death. In the present study we investigated calcineurin dependent or independent cytotoxic effects of CsA, a calcineurin inhibitor, in cervical cancerous SiHa cells. Decreased cell viability and massive cytoplasmic vacuolations were observed in CsA treated SiHa cells, having increased calcineurin activity. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR), accompanied by a decrease in cyclophilin B (ER resident PPIase), preceded the formation of the vacuoles. These vacuoles stained positive for many ER resident markers confirming their ER origin; but the absence of autophagosomal marker, LC3II, ruled out autophagy. Extensively vacuolated cells eventually undergo cell death which lacked the typical apoptotic features, but showed significant decrease in AIP (ALG2 interacting protein) as seen in paraptosis. ER-vacuolation was prevented by cycloheximide and salubrinal thereby indicating requirement of active protein synthesis. Inhibiting calcineurin activity by either Tacrolimus (FK506) or by knockdown of calcineurin B subunit did not result in either ER-stress or cellular vacuolation. However, knockdown of cyclophilin B by siRNA resulted in increased expression of Bip and IRE1α, together with cytoplasmic vacuolation. In conclusion, we report that persistent ER stress due to cyclophilin B inhibition in CsA treated cervical cancer cells caused cellular vacuolation which culminated in a non-apoptotic cell death response similar to paraptosis. Additionally, the paraptotic effects of CsA are independent of calcineurin inhibition.     

Activated Ras induces cytoplasmic vacuolation and non-apoptotic death in glioblastoma cells via novel effector pathways

Expression of activated H-Ras induces a unique form of non-apoptotic cell death in human glioblastoma cells and other specific tumor cell lines. The major cytopathological features of this form of death are the accumulation of large phase-lucent, LAMP1-positive, cytoplasmic vacuoles. In this study we sought to determine if induction of cytoplasmic vacuolation a) depends on Ras farnesylation, b) is specific to H-Ras, and c) is mediated by signaling through the major known Ras effector pathways. We find that the unusual effects of activated H-Ras depend on farnesylation and membrane association of the GTPase. Both H-Ras(G12V) and K-Ras4B(G12V) stimulate vacuolation, but activated forms of Cdc42 and RhoA do not. Amino acid substitutions in the Ras effector domain, which are known to selectively impair its interactions with Raf kinase, class-I phosphatidylinositide 3-kinase (PI3K), or Ral nucleotide exchange factors, initially pointed to Raf as a possible mediator of cell vacuolation. However, the MEK inhibitor, PD98059, did not block the induction of vacuoles, and constitutively active Raf-Caax did not mimic the effects of Ras(G12V). Introduction of normal PTEN together with H-Ras(G12V) into U251 glioblastoma cells reduced the PI3K-dependent activation of Akt, but had no effect on vacuolation. Finally, co-expression of H-Ras(G12V) with a dominant-negative form of RalA did not suppress vacuolation. Taken together, the observations indicate that Ras activates non-conventional and perhaps unique effector pathways to induce cytoplasmic vacuolation in glioblastoma cells. Identification of the relevant signaling pathways may uncover specific molecular targets that can be manipulated to activate non-apoptotic cell death in this type of cancer.     

Proteasomal dysfunction and ER stress triggers 2′-hydroxy-retrochalcone-induced paraptosis in cancer cells

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2′-hydroxy-retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin-like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin-like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy.     

WIN55,212-2 induces cytoplasmic vacuolation in apoptosis-resistant MCL cells

Cannabinoid receptors 1 (CB1) and/or 2 (CB2) are overexpressed in many types of human malignancies including mantle cell lymphoma (MCL). Agonists to CB1 and CB2 promote ceramide de novo synthesis, p38–mitogen-activated protein kinase-dependent activation of caspase-3 and apoptotic cell death in most MCLs. However, in this report we describe that in some MCLs the response to treatment with cannabinoids decreased cell viability as assessed by metabolic activity but did not involve the caspase-3 cascade or loss of plasma membrane integrity. Both primary cells from one MCL patient and the MCL cell line Granta519 responded to treatment with cannabinoids by formation of cycloheximide-sensitive cytoplasmic vacuoles, but did not enter apoptosis. The persistent expression of mammalian homolog of Atg8 with microtubule-associated protein-1 light chain-3 II (LC3 II) and p62, as well as the lack of protection from chloroquine, indicates that lysosomal degradation is not involved in this cytoplasmic vacuolation process, distinguishing from classical autophagy. Transmission electron microscopy images and immunofluorescence staining of endoplasmic reticulum (ER) chaperone calreticulin showed that the vacuoles were of ER origin and that chromatin remained normal. These features resemble paraptosis-like cell death—a third type of a programmed cell death not previously described in response to cannabinoids.     

Purinergic Receptor-mediated Rapid Depletion of Nuclear Phosphorylated Akt Depends on Pleckstrin Homology Domain Leucine-rich Repeat Phosphatase,Calcineurin, Protein Phosphatase 2A,and PTEN Phosphatases

Akt is an important oncoprotein, and data suggest a critical role for nuclear Akt in cancer development. We have previously described a rapid (3–5 min) and P2X7-dependent depletion of nuclear phosphorylated Akt (pAkt) and effects on downstream targets, and here we studied mechanisms behind the pAkt depletion. We show that cholesterol-lowering drugs, statins, or extracellular ATP, induced a complex and coordinated response in insulin-stimulated A549 cells leading to depletion of nuclear pAkt. It involved protein/lipid phosphatases PTEN, pleckstrin homology domain leucine-rich repeat phosphatase (PHLPP1 and -2), protein phosphatase 2A (PP2A), and calcineurin. We employed immunocytology, immunoprecipitation, and proximity ligation assay techniques and show that PHLPP and calcineurin translocated to the nucleus and formed complexes with Akt within 3 min. Also PTEN translocated to the nucleus and then co-localized with pAkt close to the nuclear membrane. An inhibitor of the scaffolding immunophilin FK506-binding protein 51 (FKBP51) and calcineurin, FK506, prevented depletion of nuclear pAkt. Furthermore, okadaic acid, an inhibitor of PP2A, prevented the nuclear pAkt depletion. Chemical inhibition and siRNA indicated that PHLPP, PP2A, and PTEN were required for a robust depletion of nuclear pAkt, and in prostate cancer cells lacking PTEN, transfection of PTEN restored the statin-induced pAkt depletion. The activation of protein and lipid phosphatases was paralleled by a rapid proliferating cell nuclear antigen (PCNA) translocation to the nucleus, a PCNA-p21^cip1 complex formation, and cyclin D1 degradation. We conclude that these effects reflect a signaling pathway for rapid depletion of pAkt that may stop the cell cycle.     

Superoxide anion and proteasomal dysfunction contribute to curcumin-induced paraptosis of malignant breast cancer cells

Curcumin is considered a pharmacologically safe agent that may be useful in cancer chemoprevention and therapy. Here, we show for the first time that curcumin effectively induces paraptosis in malignant breast cancer cell lines, including MDA-MB-435S, MDA-MB-231, and Hs578T cells, by promoting vacuolation that results from swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). Inhibition of protein synthesis by cycloheximide blocked curcumin-induced vacuolation and subsequent cell death, indicating that protein synthesis is required for this process. The levels of AIP-1/Alix protein, a known inhibitor protein of paraptosis, were progressively downregulated in curcumin-treated malignant breast cancer cells, and AIP-1/Alix overexpression attenuated curcumin-induced death in these cells. ERK2 and JNK activation were positively associated with curcumin-induced cell death. Mitochondrial superoxide was shown to act as a critical early signal in curcumin-induced paraptosis, whereas proteasomal dysfunction was mainly responsible for the paraptotic changes associated with ER dilation. Notably, curcumin-induced paraptotic events were not observed in normal breast cells, including mammary epithelial cells and MCF-10A cells. Taken together, our findings on curcumin-induced paraptosis may provide novel insights into the mechanisms underlying the selective anti-cancer effects of curcumin against malignant cancer cells.     

Non-Specific Blocking of miR-17-5p Guide Strand in Triple Negative Breast Cancer Cells by Amplifying Passenger Strand Activity

Conventional wisdom holds that only one of the two strands in a micro ribonucleic acid (miRNA) precursor duplex is selected as the active miRNA guide strand. The complementary miRNA passenger strand, however, is thought to be inactive. High levels of the oncogenic miRNA (oncomiR) guide strand called miR-17-5p is overexpressed in triple negative breast cancer (TNBC) and can inhibit ribosomal translation of tumor suppressor gene mRNAs, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, antisense knockdown of oncomiR miR-17-5p guide strand reduced PDCD4 and PTEN proteins by 1.8±0.3 fold in human TNBC cells instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3’UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knock down miR-17-5p reduced PDCD4 and PTEN protein expression instead of raising them. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand.     

Bcl2 at the endoplasmic reticulum protects against a Bax/Bak-independent paraptosis-like cell death pathway initiated via p20Bap31

Bap31 is an integral ER membrane protein which functions as an escort factor in the sorting of newly synthesized membrane proteins within the endoplasmic reticulum (ER). During apoptosis signaling, Bap31 is subject to early cleavage by initiator caspase-8. The resulting p20Bap31 (p20) fragment has been shown to initiate proapoptotic ER-mitochondria Ca2+ transmission, and to exert dominant negative (DN) effects on ER protein trafficking. We now report that ectopic expression of p20 in E1A/DNp53-transformed baby mouse kidney epithelial cells initiates a non-apoptotic form of cell death with paraptosis-like morphology. This pathway was characterized by an early rise in ER Ca2+ stores and massive dilation of the ER/nuclear envelope, dependent on intact ER Ca2+ stores. Ablation of the Bax/Bak genes had no effect on these ER/nuclear envelope transformations, and delayed but did not prevent cell death. ER-restricted expression of Bcl2 in the absence of Bax/Bak, however, delayed both ER/nuclear envelope dilation and cell death. This prosurvival role of Bcl2 at the ER thus extended beyond inhibition of Bax/Bak, and correlated with its ability to lower ER Ca2+ stores. Furthermore, these results indicate that ER restricted Bcl2 is capable of antagonizing not only apoptosis, but also a non-apoptotic, Bax/Bak independent, paraptosis-like form of cell death.     

Statin induces inhibition of triple negative breast cancer (TNBC) cells via PI3K pathway

Primary TNBCs are treated as if they were a single disease entity, yet it is clear they do not behave as a single entity in response to current therapies. Recently, we reported that statins might have a potential benefit for TNBCs associated with ets-1 overexpression. The aim of this study is to investigate the role of PTEN loss in the effects of statin on TNBC cells. In addition, we analyze the relationship between AKT downstream pathways and the effects of statin on TNBC cells. We investigated the effect of a statin on TNBC cells and analyzed the association of PI3K pathways using various TNBC cells in terms of PTEN loss and AKT pathways. Simvastatin treatments resulted in decreased cell viabilities in various TNBC cell lines. Compared with PTEN wild-type TNBC cells, PTEN mutant-type TNBC cells showed a decreased response to simvastatin. Expressions of phosphorylated Akt and total Akt showed an inverse relationship with PTEN expression. The TNBC cell lines, which showed increased expression of p-Akt, appeared to attenuate the expression of p-Akt by PTEN loss in simvastatin-treated TNBC cells. The Akt inhibitor, LY294002, augmented the effect of simvastatin on PTEN wild-type TNBC cells. Simvastatin induces inhibition of TNBC cells via PI3K pathway activation.     

Metabotropic Glutamate Receptor-1 Contributes to Progression in Triple Negative Breast Cancer

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy.     

Platycodin D,a bioactive component of Platycodon grandiflorum,induces cancer cell death associated with extreme vacuolation

Platycodin D (PD) is a major active component of the roots of Platycodon grandiflorum (Jacq.) A.DC. and possesses multiple biological and pharmacological properties, including anti-cancer activity. The aim of this study was to characterize PD-induced cytoplasmic vacuolation in human cancer cells and investigate the underlying mechanisms. PD-induced cancer cell death was associated with cytoplasmic pinocytic and autophagic vacuolation. Cellular energy levels were decreased by this compound, leading to the activation of AMP-activated protein kinase (AMPK). Additionally, compound C, an inhibitor of AMPK, completely prevented PD-induced vacuolation. These results suggest that PD induces cancer cell death, associated with excessive vacuolation through AMPK activation when cellular energy levels are low. Therefore, our findings provide a mechanistic rationale for a novel combinatorial approach using PD to treat cancer.     

Alternative HER/PTEN/Akt pathway activation in HPV positive and negative penile carcinomas

Background

The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood, though risk factors include human papillomavirus (HPV). Disruption of HER/PTEN/Akt pathway is present in many cancers; however there is little information on its function in PSCC. We investigated HER family receptors and phosphatase and tension homolog (PTEN) in HPV-positive and negative PSCC and its impact on Akt activation using immunohistochemistry and fluorescent in situ hybridisation (FISH).

Methodology/Principal Findings

148 PSCCs were microarrayed and immunostained for phosphorylated EGFR (pEGFR), HER2, HER3, HER4, phosphorylated Akt (pAkt), Akt1 and PTEN proteins. EGFR and PTEN gene status were also evaluated using FISH. HPV presence was assessed by PCR. pEGFR expression was detected significantly less frequently in HPV-positive than HPV-negative tumours (p = 0.0143). Conversely, HER3 expression was significantly more common in HPV-positive cases (p = 0.0128). HER4, pAkt, Akt and PTEN protein expression were not related to HPV. HER3 (p = 0.0054) and HER4 (p = 0.0002) receptors significantly correlated with cytoplasmic Akt1 immunostaining. All three proteins positively correlated with tumour grade (HER3, p = 0.0029; HER4, p = 0.0118; Akt1, p = 0.0001). pEGFR expression correlated with pAkt but not with tumour grade or stage. There was no EGFR gene amplification. HER2 was not detected. PTEN protein expression was reduced or absent in 62% of tumours but PTEN gene copy loss was present only in 4% of PSCCs.

Conclusions/Significance

EGFR, HER3 and HER4 but not HER2 are associated with penile carcinogenesis. HPV-negative tumours tend to express significantly more pEGFR than HPV-positive cancers and this expression correlates with pAkt protein, indicating EGFR as an upstream regulator of Akt signalling in PSCC. Conversely, HER3 expression is significantly more common in HPV-positive cases and positively correlates with cytoplasmic Akt1 expression. HER4 and PTEN protein expression are not related to HPV infection. Our results suggest that PSCC patients could benefit from therapies developed to target HER receptors.     

Ursolic Acid Attenuates Diabetic Mesangial Cell Injury through the Up-Regulation of Autophagy via miRNA-21/PTEN/Akt/mTOR Suppression

ObjectiveTo investigate the effect of ursolic acid on autophagy mediated through the miRNA-21-targeted phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in rat mesangial cells cultured under high glucose (HG) conditions.MethodsRat glomerular mesangial cells were cultured under normal glucose, HG, HG with the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or HG with ursolic acid conditions. Cell proliferation and hypertrophy were assayed using an MTT assay and the ratio of total protein to cell number, respectively. The miRNA-21 expression was detected using RT-qPCR. The expression of phosphatase and tensin homolog (PTEN)/AKT/mTOR signaling signatures, autophagy-associated protein and collagen I was detected by western blotting and RT-qPCR. Autophagosomes were observed using electron microscopy.ResultsCompared with mesangial cells cultured under normal glucose conditions, the cells exposed to HG showed up-regulated miRNA-21 expression, down-regulated PTEN protein and mRNA expression, up-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and down-regulated LC3II expression. Ursolic acid and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation, down-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and up-regulated LC3II expression. However, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not affect the expression of miRNA-21 and PTEN. Ursolic acid down-regulated miRNA-21 expression and up-regulated PTEN protein and mRNA expression.ConclusionsUrsolic acid inhibits the glucose-induced up-regulation of mesangial cell miRNA-21 expression, up-regulates PTEN expression, inhibits the activation of PI3K/Akt/mTOR signaling pathway, and enhances autophagy to reduce the accumulation of the extracellular matrix and ameliorate cell hypertrophy and proliferation.     

The hsp70 inhibitor VER155008 induces paraptosis requiring de novo protein synthesis in anaplastic thyroid carcinoma cells

In this study, we evaluated the effect of the hsp70 inhibitor VER155008 on survival of anaplastic thyroid carcinoma (ATC) cells. In ATC cells, VER155008 increased the percentages of dead cells and vacuolated cells. VER155008 did not lead to the cleavage of caspase-3 protein regardless of pretreatment with z-VAD-fmk. VER155008 increased LC3-II protein levels but the protein levels were not changed by autophagy inhibitors. VER155008 caused the dilatation of endoplasmic reticulum (ER), and the increased mRNA levels of Bip and CHOP, suggesting paraptosis. VER155008-induced paraptosis was attenuated by pretreatment with cycloheximide. In conclusion, VER155008 induces paraptosis characterized by cytoplasmic vacuolation, independence of caspase, dilatation of ER and induction of ER stress markers in ATC cells. Moreover, VER155008-induced paraptosis requires de novo protein synthesis in ATC cells.     

Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells

We previously reported that marchantin M (Mar) is an active agent to induce apoptosis in human prostate cancer (PCa), but the molecular mechanisms of action remain largely unknown. Here, we demonstrate that Mar potently inhibited chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of 20S proteasome both in in vitro and intracellular systems and significantly induced the accumulation of polyubiquitinated proteins in PCa cells. The computational modeling analysis suggested that Mar non-covalently bound to active sites of proteasome β5 and β1 subunits, resulting in a non-competitive inhibition. Proteasome inhibition by Mar subsequently resulted in endoplasmic reticulum (ER) stress, as evidenced by elevated glucose-regulated protein 78 and CHOP, increased phospho-eukaryotic translation initiation factor 2α (eIF_2α), splicing of X-box-binding protein-1 and dilation of the ER. However, Mar-mediated cell death was not completely impaired by a pan inhibitor of caspases. Further studies revealed that the Mar-induced cell death was greatly associated with the activation of autophagy, as indicated by the significant induction of microtubule-associated protein-1 light chain-3 beta (LC3B) expression and conversion. Electron microscopic and green fluorescent protein-tagged LC3B analyses further demonstrated the ability of autophagy induction by Mar. Time kinetic studies revealed that Mar induced a rapid and highly sustained processing of LC3B in treated cells and simultaneously decreased the expression of p62/SQSTM1. Pharmacological blockade or knockdown of LC3B and Atg5 attenuated Mar-mediated cell death. The autophagic response triggered by Mar required the activation of RNA-dependent protein kinase-like ER kinase/eIF_2α and suppression of the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin axis via preventing activation and expression of Akt. Our results identified a novel mechanism for the cytotoxic effect of Mar, which strengthens it as a potential agent in cancer chemotherapy.     

The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) represents an anomalous subset of breast cancer with a greatly reduced (30%) 5-year survival rate. The enhanced mortality and morbidity of TNBC arises from the high metastatic rate, which requires the acquisition of AnR, a process whereby anchorage-dependent cells become resistant to cell death induced by detachment. In this study TNBC cell lines were selected for AnR, and these cell lines demonstrated dramatic enhancement in the formation of lung metastases as compared with parental cells. Genetic analysis of the AnR subclones versus parental cells via next generation sequencing and analysis of global alternative RNA splicing identified that the mRNA splicing of cytoplasmic polyadenylation element binding 2 (CPEB2), a translational regulator, was altered in AnR TNBC cells. Specifically, increased inclusion of exon 4 into the mature mRNA to produce the CPEB2B isoform was observed in AnR cell lines. Molecular manipulations of CPEB2 splice variants demonstrated a key role for this RNA splicing event in the resistance of cells to anoikis. Specifically, down-regulation of the CPEB2B isoform using siRNA re-sensitized the AnR cell lines to detachment-induced cell death. The ectopic expression of CPEB2B in parental TNBC cell lines induced AnR and dramatically increased metastatic potential. Importantly, alterations in the alternative splicing of CPEB2 were also observed in human TNBC and additional subtypes of human breast cancer tumors linked to a high metastatic rate. Our findings demonstrate that the regulation of CPEB2 mRNA splicing is a key mechanism in AnR and a driving force in TNBC metastasis.     

Silencing p110β prevents rapid depletion of nuclear pAkt

The p110β subunit in the class IA PI3K family may act as an oncogene and is critical for prostate tumor development in PTEN knockout mice. We tested the possible involvement of p110β in a recently described rapid depletion of phosphorylated Akt (pAkt) in the nucleus. Previous work showed that this down-regulation is induced by extracellular ATP or by statins and is mediated by the purinergic receptor P2X7. Here, we used p110β knock out mouse embryonic fibroblasts (MEFs) and siRNA-treated cancer cells. We found that p110β is essential for ATP- or statin-induced nuclear pAkt depletion in MEFs and in several cancer cell lines including prostate cancer cells. ATP, statin or the selective P2X7 agonist BzATP also inhibited cell growth, and this inhibition was not seen in p110β knock out cells. We also found that p110β was necessary for statin-induced changes in binding between FKBP51, pAkt and PTEN. Our data show that p110β is essential for the ATP- and statin-induced effects and support a role of nuclear pAkt in cancer development. They also provide support for a chemopreventive effect of statins mediated by depletion of nuclear pAkt.     

Autophagic cell death induced by TrkA receptor activation in human glioblastoma cells

The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death.